Hi Vanessa, I think this is interesting. I just got a few questions. First, is the binding site biochemically defined, for example, by using purified proteins? Did you ChIP to see if your TF still binds to the gene after CRISPR? I think it's important to show by ChIP whether your TF still binds before you can consider other possible explanations.

I already tried a ChIP after CRISPR but unfortunately it did not as good as last time and it is hard to compare it to the parental, non-CRISPR cells.. I found the binding site by checking the DNA sequence and UCSC browser..

Two possibilities come to mind. First, perhaps you did remove the binding site in one of the alleles (but not both) and cannot distinguish when screening, in this case you would then expect to half the amount of your TF binding (depending on your sensitivity you may not be able to differentiate the two conditions). Second, and perhaps more likely, your TF has some cooperative binding with another factor binding downstream. Disruption of this downstream site might lead to much weaker binding of your TF to your binding site.

Hope it helps,

alvaro

Dear Alvaro, thanks for your help. Since I sequenced several single clones of my CRISPR cell line I know it’s homogenous. I was also thinking of either co-binding with other TFs or disrupting the DNA structure that bad, that my TF is not binding anymore.. but that’s hard to prove..

You can always check if the CRISPR deleted sequence contains some known TF / DNA interacting protein binding site or look in the literature if your TF is known to interact with some other cofactor. Maybe can you try some EMSA with your amplified WT and CRISPR binding sequence to show that your TF doesn't bind any more after CRISPR editing? A working ChIP-qPCR might be good also, particularly if you have other target genes which are not modified by your CRISPR. Do you get any modification in the targeted gene expression after CRISPR? It is also a good functional output...

If you want to definitely prove whether your protein still binds to the DNA or not, than you have to demonstrate this by CHIP analysis, ideally run in wt and CRISPR-modified cell lines in parallel. When you write " CHIP... did not as good as last time" it sounds for me as if this experiment isn't robust enough to draw any conclusion (even the observed binding in wt cells might be an artefact). So may be you should reconsider the specificity of the binding at all.

You can try to map the binding site exactly with DNase footprinting assay or run EMSA assay. Alternatively, you can order biotin-labeled DNA fragments containing your binding site or any deletion/mutation of it and run "pull-down" binding assay with your TF protein.

Here is an example of how it can work:

Article The CHR promoter element controls cell cycle-dependent gene ...

Another point is your conclusion from "I see no more downstream effects in my cells which means that my TF didnt bind anymore". This is an over-interpretation which is not necessarily true. Indeed, when you delete by CRISPR the DNA sequence in the gene promoter region downstream of your supposed binding site this can affect the expression of your target gene and cause some downstream effects regardless of your TF, in case this destroyed DNA region itself is essential for gene activation.